Characterization of human Treg-induced senescent T cells. (A) CD27 and CD28, but not other markers in naive CD4⁺ T cells, were decreased significantly by treatment with CD4⁺CD25^hiFoxP3⁺ Treg cells. The expression markers on Treg-treated T cells were determined by the FACS analysis. (B) Cytokine profile of Treg-induced senescent CD4⁺ T cells. CD4⁺ T cells treated with CD4⁺CD25⁻ effector T cells served as a control. Treated CD4⁺ T cells were purified and cytokine secretion evaluated using ELISA. Cytokine concentrations (pg/mL) are shown as the means ± SD from 3 independent experiments. (C) Suppressive function of Treg-induced senescent T cells. Both senescent CD4⁺ and CD8⁺ T cells induced by CD4⁺CD25^hiFoxp3⁺ Treg cells inhibited the proliferation of responding CD4⁺ T cells strongly. In contrast, naive T cells treated or untreated with control CD4⁺CD25⁻ effector T cells did not affect the proliferation of responding CD4⁺ T cells. CFSE-labeled naive CD4⁺ and CD8⁺ T cells were cocultured with CD4⁺CD25^hiFoxP3⁺ Treg cells or control CD4⁺CD25⁻ effector T cells for 3 days. Treated CD4⁺ and CD8⁺ T cells were purified and the suppressive activities on CD4⁺ T-cell proliferation were evaluated using [³H]-thymidine incorporation assays. Data are representative of 3 independent experiments with similar results. (D) Neutralizing Abs against IL-10 and TGF-β failed to block the suppressive activity mediated by Treg-induced senescent T cells. The suppression mediated by senescent T cells on the proliferative activity of additional naive CD4⁺ T cells was determined using [³H]-thymidine incorporation assays in the presence of mouse anti–human neutralizing Abs against IL-10 and TGF-β. Results shown are means ± SD from 3 independent experiments.